The novel metallo-β-lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis

Metallo-β-lactamases (MBLs) may hydrolyze the β-lactam rings of antibiotics. PNGM-1 is a subclass B3 deep-sea sediment MBL that predates the antibiotic era. Crystals of native and selenomethionine-substituted PNGM-1 diffracted to 2.1 and 2.3 Å resolution, respectively. They belonged to space group P2₁ and probably contain 6–10 molecules in the crystallographic asymmetric unit.

Abstract

Metallo-β-lactamases (MBLs) are present in major Gram-negative pathogens and environmental species, and pose great health risks because of their ability to hydrolyze the β-lactam rings of antibiotics such as carbapenems. PNGM-1 was the first reported case of a subclass B3 MBL protein that was identified from a metagenomic library from deep-sea sediments that predate the antibiotic era. In this study, PNGM-1 was overexpressed, purified and crystallized. Crystals of native and selenomethionine-substituted PNGM-1 diffracted to 2.10 and 2.30 Å resolution, respectively. Both the native and the selenomethionine-labelled PNGM-1 crystals belonged to the monoclinic space group P2₁, with unit-cell parameters a = 122, b = 83, c = 163 Å, β = 110°. Matthews coefficient (V _M) calculations suggested the presence of 6–10 molecules in the asymmetric unit, corresponding to a solvent content of ∼31–58%. Structure determination is currently in progress.

1. Introduction  

Metallo-β-lactamases (MBLs) are bacterial enzymes that can catalyze the hydrolysis of nearly all β-lactam antibiotics except aztreonam and require zinc or another heavy metal for catalysis (Palzkill, 2013 ▸). They are classified as molecular class B according to Ambler (1980 ▸) and as group 3 according to the Bush–Jacoby–Medeiros functional classification (Bush et al., 1995 ▸), and are divided into three subclasses, namely B1, B2 and B3, on the basis of primary amino-acid sequence homology and metal requirement (Galleni et al., 2001 ▸). Subclass B1 and B3 MBLs require two zinc ions for maximal enzyme activity, whereas subclass B2 MBLs require one bound zinc ion for maximal activity (Garau et al., 2004 ▸). The zinc-binding motifs, which include six residues at the active site, are conserved among all three subclasses (Galleni et al., 2001 ▸). The most common families of acquired class B MBLs identified in Entero­bacteriaceae include the VIM, IMP and NDM groups that belong to subclass B1 (Nordmann et al., 2011 ▸; Walsh et al., 2005 ▸; Yong et al., 2009 ▸). Subclass B3 MBLs derived from environmental bacteria, including L1 from Stenotrophomonas maltophilia (Walsh et al., 1994 ▸), FEZ-1 from Legionella gormanii (Boschi et al., 2000 ▸), GOM-1 from Chryseobacterium meningosepticum (Bellais et al., 2000 ▸), THIN-B from Janthinobacterium lividum (Rossolini et al., 2001 ▸), CAU-1 from Caulobacter crescentus (Docquier et al., 2002 ▸), CAR-1 from Erwinia carotovora (Stoczko et al., 2008 ▸) and POM-1 from Pseudomonas otitidis (Thaller et al., 2011 ▸), have been reported. In particular, the novel subclass B3 MBLs Rm3, LRA-2, LRA-3, LRA-7, LRA-8, LRA-9, LRA-12, LRA-17, LRA-19, CRD3-1, GRD33-1, ALG6-1, ALG11-1 and DHT2-1 have recently been identified in a meta­genomic library from environmental samples (Salimraj et al., 2016 ▸; Gudeta et al., 2016 ▸; Donato et al., 2010 ▸; Allen et al., 2009 ▸). Crystal structures have been determined of subclass B3 MBLs from environmental bacteria or samples: L1 (Ullah et al., 1998 ▸), FEZ-1 (García-Sáez et al., 2003 ▸) and Rm3 (Salimraj et al., 2016 ▸). Compared with the subclass B1 enzymes, the subclass B3 MBLs have been less well studied (Salimraj et al., 2016 ▸). Thus, the analysis of subclass B3 MBLs from environmental sources should expand our knowledge of their activity and structure.

A functional metagenomic library from the deep-sea sediments of Edison Seamount in Papua New Guinea, which comprised 81 100 fosmid clones, has previously been constructed (Jeon et al., 2011 ▸). We recently reported a novel subclass B3 MBL, PNGM-1, derived from a functional metagenomic library from deep-sea sediments that predate the antibiotic era (Park et al., 2018 ▸). Minimum inhibitory concentrations determined using Escherichia coli TOP10 cells harbouring the bla _PNGM-1 gene indicated a reduced susceptibility to penicillins, narrow- and extended-spectrum cephalo­sporins, and carbapenems (Park et al., 2018 ▸). Additionally, kinetic analyses showed that PNGM-1 hydrolyzed almost all β-lactams (Park et al., 2018 ▸). In this study, we report the characterization, crystallization and preliminary crystallo­graphic analysis of PNGM-1 from a functional metagenomic library from deep-sea sediments of Edison Seamount.

2. Materials and methods  

2.1. Plasmid construction  

The bla _PNGM-1 gene (GenBank ID MF445022) was subcloned into the pHSG398 vector (Takara, Kyoto, Japan) from the fosmid library of deep-sea sediments as described previously (Park et al., 2018 ▸). The assembly of a pET-28a(+) vector (Novagen, Madison, Wisconsin, USA) encoding N-terminally His₆-tagged PNGM-1 has been described previously (Park et al., 2018 ▸). Macromolecule-production information is summarized in Table 1 ▸.

Table 1. Macromolecule-production information.

Source organism	Environmental DNA
Expression vector	pET-28a(+) (Novagen)
Expression host	E. coli BL21 (DE3)
Complete amino-acid sequence of the construct produced	MHHHHHHDDDDKAGGKVTSSTGIAPKRYVYYPGSEELGPDEIRVIACGTGMPTARRAQAAAAWVVELGNGDKFIVDIGSGSMANIQSLMIPANYLTKIFLTHLHTDHWGDLVSMWAGGWTAGRTDPLEVWGPSGSREDMGTKYAVEHMLKAYNWDYMTRAVTINPRPGDINVHEFDYRALNEVVYQENGVTFRSWPCIHAGDGPVSFALEWNGYKVVFGGDTAPNIWYPEYAKGADLAIHECWMTSDQMMTKYNQPAQLALRINLDFHTSAQSFGQIMNMVQPRHAVAYHFFNDDDTRYDIYTGVRENYAGPLSMATDMMVWNITRDAVTERMAVSPDHAWDVAGPSEDLAPDRNRASEYTQYILDGRLNVDEANAHWKQEFMGRTGLTTEDLGVGS

2.2. Expression and purification of PNGM-1  

E. coli BL21 (DE3) cells harbouring pET-28a(+)/His₆-PNGM-1 were grown in LB medium containing 50 mg ml⁻¹ kanamycin to an OD_600 nm of 0.6 at 303 K; 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was then added to the culture to induce protein expression. After 16 h of cultivation at 289 K, the cells were harvested by centrifugation at 4700g for 10 min at 277 K and resuspended in ice-cold 20 mM sodium phosphate pH 7.0. The cells were disrupted by sonication and centrifuged at 20 000g for 60 min at 277 K. The clarified supernatant was loaded onto a His-Bind column (Novagen, Wisconsin, USA) that had been equilibrated with binding buffer (20 mM sodium phosphate pH 7.9, 10 mM imidazole, 500 mM NaCl). His₆-PNGM-1 was eluted with the same buffer containing 500 mM imidazole. For further purification, the His₆ tag was removed from His₆-PNGM-1 using enterokinase according to the manufacturer’s instructions (Novagen). The reaction mixture was desalted and concentrated using a Fast Desalting column (Amersham Biosciences, UK) and was then loaded onto a Mono S column (Amersham Biosciences) that had been pre-equilibrated with 10 mM sodium phosphate pH 7.0. PNGM-1 was eluted with a linear gradient of NaCl (0–0.5 M) in phosphate buffer, followed by size-exclusion chromatography on a Superdex 200 (16/60) column (GE Healthcare) equilibrated with 10 mM MES pH 6.8 at a flow rate of 1 ml min^–1. The protein concentration was measured using the Bio-Rad protein-assay kit (Bio-Rad, Hercules, California, USA) with bovine serum albumin as a standard (Bradford, 1976 ▸). The protein purity was assessed by SDS–PAGE under denaturing conditions as described by Laemmli (1970 ▸).

2.3. Biochemical characterization  

Isoelectric focusing (IEF) analysis was performed using an ampholine polyacrylamide gel with a pH range of 3–10 (Ready Gel IEF Precast Gels; Bio-Rad) for 150 min, followed by the stepwise application of 100 V (60 min), 250 V (60 min) and a final step of 500 V (30 min) using a PowerPac HV High-Voltage Power Supply (Bio-Rad, Hercules, California, USA). To detect the β-lactam hydrolytic activity of PNGM-1, an overlay assay using 100 mM nitrocefin solution (Oxoid, Basingstoke, England; Walsh et al., 1996 ▸) was performed. The pI of PNGM-1 was determined using a pH 3–10 broad-range pI calibration kit (Serva Electrophoresis GmbH, Heidelberg, Germany).

2.4. Crystallization  

Crystallization conditions for native and selenomethionine (SeMet)-labelled PNGM-1 were initially screened using the sitting-drop vapour-diffusion method with a Hydra II eDrop automated pipetting system (Matrix Technologies, Stafford, England) at 293 K. The initial crystallization conditions were obtained using the Wizard Classic and Wizard Precipitant Synergy kits (Rigaku Reagents, Bainbridge Island, Washington, USA). The drops consisted of 0.5 µl protein solution and 0.5 µl reservoir solution and were equilibrated against 50 µl reservoir solution. After two weeks, native PNGM-1 crystals were observed using Wizard Classic 2 condition No. 43 [0.1 M Tris–HCl pH 7.0, 0.2 M MgCl₂, 10%(w/v) PEG 8000]. A streak-seeding technique was used to improve the quality of the native PNGM-1 crystals: the crystals were improved by streak-seeding with reservoir solution consisting of 0.1 M Tris–HCl pH 7.0, 0.2 M MgCl₂, 9%(w/v) PEG 8000. Crystals of SeMet-labelled PNGM-1 were obtained using Wizard Precipitant Synergy condition No. 147 [0.1 M HEPES pH 7.5, 0.1 M CaCl₂, 8.25%(w/v) PEG 3350, 1.32%(v/v) 2-propanol]. The native and the SeMet-labelled PNGM-1 crystals were cryo­protected in reservoir solution supplemented with 20%(v/v) 2-methyl-2,4-pentanediol. The crystals were mounted in a loop and transferred to reservoir solution for 1 min before cooling in liquid nitrogen. The cryoprotected crystals were then mounted on a goniometer in a stream of cold nitrogen gas at 100 K. Crystallization information is provided in Table 2 ▸.

Table 2. Crystallization.

	Native PNGM-1	SeMet-labelled PNGM-1
Method	Sitting-drop vapour diffusion for initial screening, hanging-drop vapour diffusion for crystal optimization	Sitting-drop vapour diffusion
Plate type	96-well plate for initial crystal screening, 24-well plates for crystal optimization	96-well plate
Temperature (K)	293	293
Protein concentration (mg ml–1)	6	6
Buffer composition of protein solution	10 mM MES pH 6.8	10 mM MES pH 6.8
Composition of reservoir solution	0.1 M Tris–HCl pH 7.0, 0.2 M MgCl2, 9%(w/v) PEG 8000	0.1 M HEPES pH 7.5, 0.1 M CaCl2, 8.25%(w/v) PEG 3350, 1.32%(v/v) 2-propanol
Volume and ratio of drop	0.5 µl, 1:1 for initial screening; 1 µl, 1:1 for crystal optimization	0.5 µl, 1:1
Volume of reservoir (µl)	50 µl for initial screening, 1000 µl for crystal optimization	50 µl for initial screening

2.5. X-ray data collection and data processing  

X-ray diffraction data were collected at 100 K using an ADSC Q315r detector on beamline 5C and an ADSC Q270 detector on beamline 7A at Pohang Light Source (PLS), Republic of Korea. X-ray diffraction data were collected to 2.10 Å resolution for native PNGM-1. The native PNGM-1 data were collected with 1° oscillation per frame to obtain 360° of data. Single-wavelength anomalous diffraction experiments were performed using SeMet-labelled PNGM-1 crystals at a wavelength of 0.97935 Å (the Se peak). SeMet-labelled PNGM-1 crystals were oscillated by 0.5° per frame over a range of 720° to obtain maximum redundancy. X-ray diffraction data were collected to 2.30 Å resolution from SeMet-labelled PNGM-1. All data were integrated and scaled using DENZO and SCALEPACK (Otwinowski & Minor, 1997 ▸). Data-collection statistics are shown in Table 3 ▸.

Table 3. Data collection and processing.

Values in parentheses are for the highest resolution shell.

	Native PNGM-1	SeMet-labelled PNGM-1, peak
Diffraction source	Beamline 5C, PLS	Beamline 7A, PLS
Wavelength (Å)	0.97935	0.97935
Detector	ADSC Q315r	ADSC Q270
Space group	P21	P21
a, b, c (Å)	122.3, 83.0, 163.5	121.9, 83.1, 162.8
α, β, γ (°)	90.0, 110.6, 90.0	0.0, 110.2, 90.0
Matthews coefficient (Å3 Da−1)	1.79–2.98	1.78–2.97
Solvent content (%)	31–58	31–58
Resolution (Å)	50.00–2.10 (2.14–2.10)	50.00–2.30 (2.34–2.30)
Total No. of reflections	1318149	1018484
No. of unique reflections	175405	135186
Completeness (%)	98.2 (97.5)	98.1 (86.8)
Multiplicity	7.5 (7.4)	7.5 (6.9)
R merge † (%)	9.0 (22.7)	12.2 (34.2)
〈I/σ(I)〉	47.7 (15.5)	46.8 (11.3)

^† R _merge = , where I(hkl) is the intensity of reflection hkl, is the sum over all reflections and is the sum over i measurements of reflection hkl.

3. Results and discussion  

PNGM-1 was previously identified in a functional meta­genomic library from deep-sea sediments from Edison Seamount (Park et al., 2018 ▸). This previous study revealed that PNGM-1 comprised a single 386-amino-acid polypeptide chain. Primary amino-acid sequence analysis of PNGM-1 indicated similarity (15–18% identical residues) to almost all MBLs of subclass B3; it showed the highest similarity to the subclass B3 MBL FEZ-1 (18% identity). Sequence alignment of PNGM-1 with FEZ-1 indicated that PNGM-1 has a zinc ion-binding motif, H₁₁₆ XH₁₁₈ XD₁₂₀H₁₂₁, H₁₉₆ and H₂₆₃ (numbering is according to the BBL scheme; Galleni et al., 2001 ▸), which is only present in subclass B3 MBLs (Jeon et al., 2015 ▸; Fig. 1 ▸).

Figure 1.

Sequence alignment of PNGM-1 with the subclass B3 metallo-β-lactamases FEZ-1 (accession No. CAB96921), L1 (accession No. ABC02083) and Rm3 (accession No. WP_071766622) derived from environmental bacteria or samples. Conserved residues are shown on a black background and the secondary-structure elements of FEZ-1 (PDB entry 1k07; García-Sáez et al., 2003 ▸) are indicated above the sequences. The amino-acid sequence of PNGM-1 showed the highest percentage of identity to FEZ-1 among the subclass B3 MBLs. The amino-acid sequences were aligned using ClustalW. The conserved zinc ion-binding residues typical of subclass B3 MBLs are indicated by red arrows.

Recombinant PNGM-1 with an N-terminal His₆ tag was expressed in E. coli BL21 (DE3) and was successfully purified to homogeneity as observed using SDS–PAGE (Fig. 2 ▸). Purified PNGM-1 showed a single protein band that correlated well with the theoretical mass (43 kDa) of PNGM-1 as determined by SDS–PAGE (Fig. 2 ▸ a). To detect β-lactamase activity of PNGM-1, an overlay assay using 100 mM nitrocefin solution was conducted and β-lactamase activity of PNGM-1 with a pI of 5.1 was confirmed using the IEF gel and hydrolysis of nitrocefin (Fig. 2 ▸ b).

Figure 2.

SDS–PAGE and isoelectric focusing (IEF) and in-gel activity staining of PNGM-1 using nitrocefin. (a) SDS–PAGE analysis of protein samples during purification. From left to right, molecular-weight markers (lane 1; labelled in kDa), whole-cell extract (lane 2), soluble protein (lane 3) and purified PNGM-­1 (lane 4). (b) IEF and overlay assay of PNGM-1. Lane 1, pI marker (pH range 3–10; labelled); lane 2, silver staining after IEF of PNGM-1; lane 3, overlay activity assay with 100 mM nitrocefin solution after IEF of PNGM-1.

Native PNGM-1 crystals were obtained by mixing 0.5 µl protein solution with 0.5 µl reservoir solution consisting of 0.1 M Tris–HCl pH 7.0, 0.2 M MgCl₂, 9%(w/v) PEG 8000. The crystals grew to maximum dimensions of approximately 0.3 × 0.3 × 0.2 mm in two weeks (Fig. 3 ▸ a). X-ray diffraction data for native PNGM-1 were collected to 2.10 Å resolution on beamline 5C at PLS, Republic of Korea (Fig. 4 ▸ a). The native PNGM-1 crystals belonged to the monoclinic space group P2₁, with unit-cell parameters a = 122.3, b = 83.0, c = 163.5 Å. Matthews coefficient analysis indicated that the asymmetric unit comprised 6–10 molecules (V _M = 1.79–2.98 ų Da⁻¹), giving a solvent content of 31–58%. SeMet-labelled PNGM-1 crystals were obtained by mixing 0.5 µl protein solution with 0.5 µl reservoir solution consisting of 0.1 M HEPES pH 7.5, 0.1 M CaCl₂, 8.25%(w/v) PEG 3350, 1.32%(v/v) 2-propanol. The crystals grew to maximum dimensions of approximately 0.25 × 0.2 × 0.2 mm in two weeks (Fig. 3 ▸ b). X-ray diffraction data for SeMet-labelled PNGM-1 were collected to 2.30 Å resolution on beamline 7A at PLS (Fig. 4 ▸ b). SeMet-labelled PNGM-1 crystals also belonged to the monoclinic space group P2₁, with unit-cell parameters a = 121.9, b = 83.1, c = 162.8 Å. The asymmetric unit comprised 6–10 molecules, with a corresponding V_M of 1.78–2.97 ų Da⁻¹ and a solvent content of 31–58%. Data-collection and processing statistics are given in Table 3 ▸.

Figure 3.

Crystals of native and SeMet-labelled PNGM-1. (a) Optimized crystals of native PNGM-1 obtained using a reservoir condition consisting of 0.1 M Tris pH 7.0, 0.2 M MgCl₂, 9%(w/v) PEG 8000. The dimensions of the native PNGM-1 crystal were approximately 0.3 × 0.3 × 0.2 mm. (b) An SeMet-labelled PNGM-1 crystal obtained using Wizard Precipitant Synergy condition No. 147 [0.1 M HEPES pH 7.5, 0.1 M CaCl₂, 8.25%(w/v) PEG 3350, 1.32%(v/v) 2-­propanol]. The dimensions of the SeMet-labelled PNGM-1 crystal were approximately 0.25 × 0.2 × 0.2 mm. The scale bar represents 0.1 mm.

Figure 4.

Diffraction images of (a) native PNGM-1 and (b) SeMet-labelled PNGM-1. Diffraction resolutions are marked as circles. Expanded views of the regions in red boxes are shown in the middle (red arrows).

In conclusion, the crystallization and preliminary crystallographic analysis of a subclass B3 MBL (PNGM-1) from a metagenomic library from deep-sea sediments from Edison Seamount (about 10 000 years old; Schmidt et al., 2002 ▸) that existed prior to the antibiotic era is the first to be reported among subclass B3 MBLs. However, manual model building and further refinement are still required for structural analyses. A structural study should reveal the catalytic mechanism and structural features of PNGM-1 in the near future.

Funding Statement

This work was funded by Ministry of Science ICT and Future Planning grants NRF-2017M3A9E4078014 and NRF-2017R1A2B4002315. Korea Centers for Disease Control & Prevention grant 2017-ER5404-01.